Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity Onajite Kousin-Ezewu, Laura Azzopardi, Richard A. Parker, et al. Neurology 2014;82;2158-2164 Published Online before print May 16, 2014 DOI 10.1212/WNL.0000000000000520 This information is current as of May 16, 2014
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://www.neurology.org/content/82/24/2158.full.html
Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2014 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.
ARTICLES
Onajite Kousin-Ezewu, MRCP Laura Azzopardi, MRCP Richard A. Parker, MSc Orla Tuohy, MRCP Alastair Compston, FRCP, PhD Alasdair Coles, FRCP, PhD Joanne Jones, MRCP, PhD
Correspondence to Dr. Jones: [email protected]
Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity ABSTRACT
Objective: To test the hypothesis that accelerated peripheral blood mononuclear cell recovery after alemtuzumab treatment of multiple sclerosis is associated with recurrent disease activity and to investigate the claim that CD4 counts greater than 388.5 3 106 cells/mL at 12 months can be used to identify patients who may benefit from further treatment.
Methods: A total of 108 patients were followed for a median of 99 months post alemtuzumab. Patients were classified as active or nonactive after each cycle of treatment based on clinical relapse, increasing disability, or new T2/enhancing MRI lesions. These outcomes were correlated with CD4, CD8, CD19, CD561 NK, and monocyte counts. Results: Of 108 patients, 56 (52%) relapsed at some point during follow-up. Mean annualized relapse rate after alemtuzumab was 0.17 vs 1.67 prior to treatment (equating to a 90% reduction). Of 108 patients, 28 (26%) met the criteria for sustained accumulation of disability. Median time to the lower limit of normal for CD19, CD8, and CD4 was 3, 19.5, and 32 months, respectively. There was no significant difference in the recovery of any cell population between patients with and without disease activity or accumulation of disability after treatment.
Conclusion: This study does not support the use of cell counts as biomarkers for identifying patients at greater risk of active disease following treatment with alemtuzumab. Neurology® 2014;82:2158–2164 GLOSSARY EDSS 5 Expanded Disability Status Scale; IQR 5 interquartile range; LLN 5 lower limit of normal; MS 5 multiple sclerosis; RRMS 5 relapsing-remitting multiple sclerosis.
Editorial, page 2150 Supplemental data at Neurology.org
Alemtuzumab has proven efficacy as a treatment for relapsing-remitting multiple sclerosis (MS). In a phase 2 trial, compared with interferon b-1a, alemtuzumab reduced the risk of relapse and sustained accumulation of disability by more than 70% at 3 years, with sustained efficacy at 5 years.1,2 Two phase 3 trials (CARE-MS I and CARE-MS II) have confirmed efficacy in treatment-naive patients and established superiority over interferon b-1a in patients who continue to relapse despite first-line therapy.2,3 So alemtuzumab was licensed by the European Medicines Agency4 and is entering routine clinical practice in the European Union as a treatment for active MS. Alemtuzumab is a lymphocyte-depleting anti-CD52 monoclonal antibody. Each cycle causes profound pan-lymphocyte depletion, but the relatively infrequent dosing regimen allows reconstitution to occur. The rate and degree of recovery varies with cell type: B cells recover rapidly, whereas T-cell lymphopenia is prolonged, with CD4 and CD8 cells taking 35 and 20 months, respectively, to reach the lower limit of normal.5 During this period of immune reconstitution, 30% of individuals experience thyroid autoimmunity, and 1% develop immune thrombocytopenic purpura; in rare cases, Goodpasture syndrome, autoimmune hemolytic anemia, and autoimmune neutropenia have also been reported.6 We have recently shown that the risk of developing autoimmunity after alemtuzumab is unrelated to rate of T-cell reconstitution but rather reflects the degree to which recovery occurs by expansion of cells that have escaped From the Department of Clinical Neurosciences (O.K.-E., L.A., O.T., A. Compston, A. Coles, J.J.) and the Centre for Applied Medical Statistics, Department of Public Health and Primary Care (R.A.P.), University of Cambridge, UK. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
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depletion rather than thymopoiesis.7 A recently published report,8 however, suggested that peripheral CD4 recovery can be used to predict MS disease activity after treatment, with counts greater than 388.5 3 106 cells/mL at 12 months following therapy identifying patients who are likely to have recurrent disease activity and who may therefore benefit from further treatment. Given the clear clinical implications of this claim, we reassessed this finding in the Cambridge cohort—a larger group of patients in whom the role of alemtuzumab in relapsingremitting MS was originally evaluated and therefore provides prolonged duration of follow-up. METHODS Patients and procedures. All patients had relapsing-remitting MS (RRMS) and had participated in CAMMS223 (a phase 2 randomized controlled trial) and CAMMS 224 or SM3 (both investigator-led, open-label studies). CAMMS223 key eligibility criteria were disease onset within 3 years, at least 2 clinical relapses during the previous 2 years, and a score of 3 or less on the Expanded Disability Status Scale (EDSS). Patients were included in CAMMS 224 and SM3 if they had at least one relapse in the previous year, an EDSS score of 6.0 or less, and disease duration of less than 10 years. Subsequently, all patients entered either CAMSAFE (an investigator-led long-term observational study) or the extension phase of the CAMMS223 trial. The first patient from this cohort was treated on November 22, 1999, with the date for final collection of data January 1, 2013.
Standard protocol approvals, registrations, and patient consents. All studies were approved by a regional ethics board and institutional research committee. All patients gave written informed consent.
Clinical treatment and follow-up protocol. All patients received at least 2 elective cycles of alemtuzumab given annually, with the potential for further cycles if there was clinical or radiologic evidence of ongoing disease activity. Patients were reviewed at 1 and 3 months and then quarterly for the first 2 years after each treatment cycle. For the following 2 years, they were seen biannually and then at least annually thereafter. Patients were also seen whenever a relapse was suspected.
Outcome assessments. For participants in the CAMMS223 study, EDSS scores were determined quarterly in a blinded fashion by a neurologist who also adjudicated possible relapses. The same assessor measured the EDSS of patients in the CAMMS224 and SM3 studies, albeit less frequently. Sustained accumulation of disability was defined as an increase of 1.5 EDSS points from a baseline of 0, or an increase of $1.0 if the baseline was $1.0 confirmed over 6 months. A relapse was defined as new neurologic symptoms attributable to MS lasting .48 hours with an objective change in neurologic examination. Peripheral blood mononuclear cell phenotyping was performed at baseline and then quarterly for the first 36 months and then at least annually (including total lymphocyte count, CD4, CD8, CD19, CD56-NK, and monocyte counts). Brain MRI scans were performed in most patients with a suspicion of active disease prior to retreatment with alemtuzumab. Monthly MRI scans were performed in a subset of patients from the SM3 study. A number of clinically
inactive patients had interval MRI scans to look for subclinical activity and to provide a means for comparison in case of future disease activity.
Statistical analysis. Median time for recovery to the lower limit of normal (LLN) was calculated for each cell subset. All data were categorized depending on the cycle of alemtuzumab treatment. Patients were placed into “active” or “nonactive” groups independent of when an event took place within a particular treatment cycle. Therefore, within each cycle, patients were defined as being relapse-free or relapsing, disability-free or having accumulated disability, or having reached a positive composite endpoint (defined as having relapsed, or accumulated disability, or had an active MRI scan) or a negative composite endpoint based on all 3 outcomes. A subgroup of patients (n 5 91), scanned after treatment, were classified as MRI active or nonactive. To assess differential lymphocyte reconstitution between groups, a linear mixed-effects regression method was undertaken with CD4/CD8/CD19/CD56/monocytes or total lymphocyte count as the outcome variable and relapse/disability/ active MRI/composite score and time point as explanatory variables. A quadratic term (time point squared) was also included due to the observed relationship between time point and outcome. A separate linear mixed-effects model was fitted within each cycle. A continuous autoregressive (order 1) correlation structure was assumed for all models. Model coefficients are presented with 95% confidence intervals and p values. A Fisher exact test was used to assess whether a CD4 count of 388.5 3 106/mL or greater at 12 months predicts disease activity, either clinically or radiologically. The standard 5% significance level was used throughout, and no adjustment made for multiple testing in order to avoid inflating the type II error rate. The linear mixed-effects regression method was implemented in R software8 using the nlme package.9,10 R software was also used to compute Fisher exact tests. All other analyses were performed in GraphPad PRISM (version 5.00 for Windows; www.graphpad.com).
Data were derived from 108 patients, of whom 73 (67.6%) were female. The median follow-up from first treatment was 99 months (interquartile range [IQR] 74.75– 117.25). The mean age of patients at first treatment with alemtuzumab was 32.8 years (SD 7.99). The median EDSS at baseline was 3.0 (IQR 1.5–4.75). Mean relapse frequency prior to treatment was 1.7 relapses per annum (SD 0.81).
RESULTS Study population characteristics.
Lymphocyte reconstitution. As previously reported,5,8,11
treatment with alemtuzumab led to profound panlymphocyte depletion, followed by differential recovery. CD19 lymphocytes reached the LLN most rapidly, with a median recovery time of 3 months (IQR 3–6). The intervals for CD8 and CD4 lymphocytes were median times of 19.5 (IQR 10–34.5) and 32 (IQR 21.75–41) months, respectively. Clinical outcomes. The total number of patients who experienced at least 1 relapse during the follow-up period was 56 (51.85%). The mean relapse frequency posttreatment was 0.17 relapses per annum, equating to an 89.8% reduction in the annualized relapse rate Neurology 82
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compared to pretreatment. Twenty-eight patients (25.9%) met the definition for sustained accumulation of disability. Ninety-one patients had an MRI scan: 16 individuals (17.6%) had an active scan (new T2/ enhancing lesions) at some point during the follow-up period (see tables e-1 and e-2 on the Neurology® Web site at Neurology.org for a detailed breakdown of each alemtuzumab cycle). Association of peripheral mononuclear cell subsets with disease activity. Relapse. Within each treatment cycle,
there was no difference in the number of CD4 T cells, CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those with and without clinically defined relapses (figure 1 for cycles 1-3, figure e-1 for cycle 4, and table 1; data not shown for NK cells and monocytes). Using the Fisher exact test, we found no association between a CD4 count of .388.5 cells 3 106/mL at 12 months and risk of relapse (p 5 0.28). Given the possibility that relapses within the first few months of treatment may be due to lymphocytes that have already entered the CNS,12 we looked at timing of relapses following cycle 2 (chosen as it is the most informative cycle in terms of patient numbers, number of relapses, and length of follow-up). Only 3 out of 106 patients relapsed within 2 months of treatment, Figure 1
and of these, 2 went on to have additional relapses within cycle 2, leaving only one patient who was potentially misclassified using our method. Given this, we are confident in our conclusions. MRI activity. Within each treatment cycle, there was no difference in the number of CD19 B cells, CD56 NK cells, or monocytes between those with and without active MRI scans (figure 2 for cycles 1-3, figure e-1 for cycle 4, and table 1; data not shown for NK cells and monocytes). CD4 cells (p 5 0.016) and CD8 cells (p 5 0.008) were found to be higher (on average by 0.146 and 0.125 3 109/mL cells, respectively) in the active MRI group (n 5 5) vs the inactive group (n 5 28) within treatment cycle 3. No difference was found in any other treatment cycle; indeed, in cycles 1 and 2 (the most informative periods numerically), the trend was in the opposite direction (figure 2, table 1). Using the Fisher exact test, we found an association between a CD4 cell count of .388.5 3 106/mL at 12 months and the risk of having an active MRI scan (overall p 5 0.02). However, further analysis of this result demonstrated that the difference was driven by patients within cycle 3 (p , 0.0001), with no difference observed within cycles 1 (p 5 1.0), 2 (p 5 0.91), or 4 (p 5 0.05). Disability accumulation. Within each treatment cycle, there was no difference in the number of
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without on-study relapses
Patients were defined as having relapsed (shown in red) if they developed neurologic symptoms attributable to multiple sclerosis lasting .48 hours with an objective change in neurologic examination in the absence of infection. Cell units are 3109/L. Error bars indicate SD. 2160
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Table 1
Comparison of CD41, CD81, and CD191 counts after each cycle of alemtuzumab in those with and without active disease CD4 Cycle
Relapse
MRI
Disability
Composite
CD8
Coefficient
95% CI
CD19
p Value
Coefficient
95% CI
p Value
Coefficient
95% CI
p Value
1
0.003
0.028 to 0.034
0.859
20.020
20.069 to 0.030
0.438
0.025
20.026 to 0.077
0.330
2
20.029
20.075 to 0.017
0.209
20.011
20.051 to 0.028
0.577
0.040
20.007 to 0.087
0.095
3
20.009
20.083 to 0.065
0.806
0.037
20.017 to 0.090
0.173
0.005
20.069 to 0.079
0.901
4
20.042
20.333 to 0.248
0.741
20.046
20.255 to 0.163
0.620
20.031
20.376 to 0.314
0.839
1
20.053
20.117 to 0.012
0.104
20.071
20.168 to 0.027
0.144
0.011
20.124 to 0.146
0.865
2
20.007
20.094 to 0.080
0.873
20.026
20.116 to 0.063
0.558
0.010
20.086 to 0.106
0.831
3
0.146
0.029 to 0.264
0.016
a
0.125
0.035 to 0.214
0.008
0.029
20.101 to 0.159
0.653
4
0.068
20.239 to 0.375
0.606
0.107
20.134 to 0.348
0.319
20.228
20.588 to 0.131
0.171
1
20.063
20.103 to 20.024
0.002a
20.008
20.076 to 0.060
0.813
0.042
20.027 to 0.111
0.226
2
20.041
20.099 to 0.016
0.154
20.009
20.057 to 0.039
0.709
0.005
20.055 to 0.065
0.861
3
20.028
20.114 to 0.058
0.522
20.022
20.084 to 0.040
0.473
20.001
20.087 to 0.084
0.977
4
20.204
20.546 to 0.139
0.203
20.104
20.374 to 0.166
0.392
20.054
20.508 to 0.399
0.784
1
20.019
20.047 to 0.009
0.179
20.004
20.050 to 0.041
0.860
0.013
20.033 to 0.060
0.571
2
20.028
20.074 to 0.018
0.233
0.002
20.035 to 0.039
0.923
0.021
20.025 to 0.068
0.367
3
20.022
20.095 to 0.051
0.550
0.021
20.032 to 0.074
0.432
0.004
20.069 to 0.077
0.915
4
20.285
20.548 to 20.023
0.037a
20.184
20.390 to 0.022
0.073
20.107
20.548 to 0.335
0.587
a
Abbreviation: CI 5 confidence interval. For each cycle, patients were defined as active or not based on clinical relapse, MRI activity, disability acquisition, and the composite endpoint. A linear mixed-effects regression method was used with CD41, CD81, CD191, CD561 NK cells, or monocytes as the outcome variable, and with relapse, disability, MRI activity, or composite score and time point as explanatory variables. A quadratic term (time point squared) was also included due to the observed relationship between time point and outcome. A separate linear mixed-effects model was fitted within each cycle. A continuous autoregressive (order 1) correlation structure was assumed for all models. Model coefficients are presented with 95% CIs and p values are shown for CD41, CD81, and CD191 cells (other subpopulations are reported in the text). a 5% significance level reached.
CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those with and without accumulation of disability (figure 3 for cycles 1-3, figure e-1 for cycle 4, and table 1). CD4 T cells were found to be lower in patients who accumulated disability in cycle 1 (adjusted mean difference across the cycle 0.063 3 109; p 5 0.002). No difference was found in any other treatment cycle, although the trend was in the same direction (figure 3, table 1). Composite. Within each treatment cycle, there was no difference in the number of CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those who did and did not reach the composite end point (table 1; data not shown for nonlymphocyte cell populations). Within cycle 4, CD4 T cells were found to be lower in patients who met the composite end point compared to those who did not. No difference was found in any other treatment cycle (table 1). Using a much larger cohort and more prolonged follow-up, we fail to confirm the claim that accelerated CD4 T cell recovery after treatment is a biomarker for recurrent MS disease activity following lymphocyte depletion with alemtuzumab. We also find no evidence that a CD4 T-cell count DISCUSSION
of greater than 388.5 3 106 cells/mL at 12 months has utility in selecting a group of patients who may benefit from more intensive monitoring or perhaps even prophylactic repeat dosing. There are a number of differences between our work and the previous report.8 First, our cohort is larger (108 vs 56 patients) with a longer duration of follow-up (median follow up of 99 months [IQR 74.75–117.25] vs 55 months [IQR 24–115]). Although both studies selected patients with active RRMS, baseline MS disease activity was somewhat higher in the previous study (annualized relapse rate of 2.6 SD 0.9 vs 1.7 SD 0.8); however, few of their patients experienced disease activity post alemtuzumab (probably reflecting their shorter follow-up): only 8/56 experienced a clinical relapse, with a further 4 patients showing MRI disease activity alone; this small number of data points makes the study susceptible to extreme outliers. Unlike the previous study, we did not perform routine MRI brain scans at month 24. As a consequence, analysis of MRI outcome is based on data from fewer patients (19 for cycle 1, 59 for cycle 2, 33 for cycle 3, and 10 for cycle 4); this is a limitation of our study. However, these differences do not explain why our 2 studies have reached opposite conclusions; this is best Neurology 82
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Figure 2
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without on-study active MRI scans
Patients were defined as having an active MRI scan (shown in red) if they had acquired new T2 lesions or enhancing lesions. Cell units are 3109/L. Error bars indicate SD.
explained by weaknesses in their statistical methods. First, they did not account for repeated treatments; only cell counts from the most recent alemtuzumab dose were analyzed: post cycle 2 CD4 counts for those who remain in remission were compared to post cycle 3 counts for those with active disease prompting redosing and CD4 counts post cycle 3 were then correlated with disease activity prior to cycle 3 and used to predict an event that had already occurred. This method assumes that reconstitution is identical after each round of treatment, representing a major limitation of their study. We controlled for this bias by looking at reconstitution and disease activity after and within each treatment cycle. Due to complexity of the analysis, the timing of the event within each cycle is still not accounted for. Secondly, the previous report compared mean cell counts at multiple time points using Student t tests, or Mann-Whitney U when non-normally distributed, without taking into account multiple nonindependent observations per patient (an individual’s CD4 count at month 12 is not independent of the month 9 count, and so on). Furthermore, p values were not corrected for multiple comparisons, of which there were many, so it is likely that some of the statistically significant results occurred by chance; when we repeated the analysis using our data, no p value survived correction (data not shown). 2162
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The wish to identify a biomarker for recurrent disease activity after alemtuzumab is to be welcomed. This would reduce the need to monitor patients at low risk of relapse and allow the preemptive treatment of highrisk patients. Although CD4 counts may be an attractive candidate—they are readily measurable and T cells are undoubtedly involved in disease pathogenesis—given the complex nature of the immune system, it is not surprising that peripheral CD4 counts alone do not predict CNS inflammation (indeed, it is known that selective anti-CD4-depleting therapies do not suppress disease activity in MS13). Also, after alemtuzumab treatment, composition of the circulating immune repertoire is radically altered. For example, for at least 6 months following each cycle, the CD4 T-cell pool is dominated by memory cells, particularly those with a regulatory phenotype (CD41CD45RA-CD35hiFoxP31IL-7Rlo).14 Self evidently, investing confidence in a single measure of a major cellular constituent of peripheral blood disregards the complexity of the immunopathogenesis of MS and is misplaced. Arguably, our data do not prove or disprove whether long-term disease stability is associated with lower CD4 counts, as patients with clinical or radiologic evidence of disease activity are automatically retreated. However, it is our position that peripheral
Figure 3
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without acquisition of disability
Patients were deemed to have met the definition of sustained accumulation of disability if their Expanded Disability Status Scale increased by $1.5 points from a baseline of 0, or by $1.0 points from a baseline of $1.0. Patients meeting this definition are shown in red. Cell units are 3109/L. Error bars indicate SD.
CD4 counts have no utility in predicting MS disease activity after alemtuzumab, and we strongly advise neurologists against using them to personalize treatment protocols. In particular, CD4 counts should not be used as a marker of the need for preemptive retreatment, thereby exposing patients to potential risk.1–3 Our cautionary message, refuting the claims of the previous report,8 is timely since alemtuzumab will soon be entering the clinic as a treatment for active RRMS. AUTHOR CONTRIBUTIONS Dr. Onajite Kousin-Ezewu designed and conducted the study, analyzed data in the study, interpreted data in the study, and drafted and revised the manuscript. Dr. Laura Azzopardi conducted the study and interpreted data in the study. Mr. Richard Parker analyzed data in the study. Dr. Orla Tuohy conducted the study. Prof Alastair Compston revised the manuscript. Dr. Alasdair J. Coles designed the study and revised the manuscript. Dr. Joanne L. Jones designed the study, analyzed data in the study, interpreted data in the study, and revised the manuscript.
STUDY FUNDING No targeted funding reported.
DISCLOSURE O. Kousin-Ezewu, L. Azzopardi, R. Parker, and O. Tuohy report no disclosures relevant to the manuscript. A. Compston reports receiving consulting fees, lecture fees, and grant support from Genzyme, and lecture fees from Bayer Schering Pharma, and has consulted for Lundbeck, all on behalf of the University of Cambridge. A. Coles reports receiving consulting and
lecture fees from Genzyme, lecture fees from Merck Serono, and research support paid to his institution from Genzyme. J. Jones reports receiving consulting fees and lecture fees from Bayer Schering Pharma and lecture fees from Genzyme. Go to Neurology.org for full disclosures.
Received August 24, 2013. Accepted in final form February 7, 2014. REFERENCES 1. Coles AJ, Compston DA, Selmaj KW, et al. Alemtuzumab vs. interferon beta-1a in early multiple sclerosis. N Engl J Med 2008;359:1786–1801. 2. Cohen JA, Coles AJ, Arnold DL, et al. Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial. Lancet 2012;380:1819–1828. 3. Coles AJ, Twyman CL, Arnold DL, et al. Alemtuzumab for patients with relapsing multiple sclerosis after diseasemodifying therapy: a randomised controlled phase 3 trial. Lancet 2012;380:1829–1839. 4. European Medicines Agency: EPAR summary for the public: Lemtrada (alemtuzumab). Available at: http://www.ema. europa.eu/docs/en_GB/document_library/EPAR_-_Summary_ for_the_public/human/003718/WC500150523.pdf. Accessed November 22, 2013. 5. Hill-Cawthorne GA, Button T, Tuohy O, et al. Long term lymphocyte reconstitution after alemtuzumab treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry 2012; 83:298–304. 6. Coles AJ, Fox E, Vladic A, et al. Alemtuzumab more effective than interferon-1a at 5-year follow-up Neurology 82
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of CAMMS223 clinical trial. Neurology 2012;78: 1069–1078. Jones JL, Thompson SA, Loh P, et al. Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation. Proc Natl Acad Sci USA 2013;110: 20200–20205. Cossburn MD, Harding K, Ingram G, et al. Clinical relevance of differential lymphocyte recovery after alemtuzumab therapy for multiple sclerosis. Neurology 2013;80:55–61. NLME: Linear and Nonlinear Mixed Effects Models [computer program]. Version R package version 3.1-109. 2013. R Core Team. R: A Language and environment for statistical computing [computer program]. Vienna, Austria: R Foundation for Statistical Computing; 2013. Available at: http://www.R-project.org/. Accessed August 16, 2013.
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Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity Onajite Kousin-Ezewu, Laura Azzopardi, Richard A. Parker, et al. Neurology 2014;82;2158-2164 Published Online before print May 16, 2014 DOI 10.1212/WNL.0000000000000520 This information is current as of May 16, 2014 Updated Information & Services
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Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2014 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.
ARTICLES
Onajite Kousin-Ezewu, MRCP Laura Azzopardi, MRCP Richard A. Parker, MSc Orla Tuohy, MRCP Alastair Compston, FRCP, PhD Alasdair Coles, FRCP, PhD Joanne Jones, MRCP, PhD
Correspondence to Dr. Jones: [email protected]
Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity ABSTRACT
Objective: To test the hypothesis that accelerated peripheral blood mononuclear cell recovery after alemtuzumab treatment of multiple sclerosis is associated with recurrent disease activity and to investigate the claim that CD4 counts greater than 388.5 3 106 cells/mL at 12 months can be used to identify patients who may benefit from further treatment.
Methods: A total of 108 patients were followed for a median of 99 months post alemtuzumab. Patients were classified as active or nonactive after each cycle of treatment based on clinical relapse, increasing disability, or new T2/enhancing MRI lesions. These outcomes were correlated with CD4, CD8, CD19, CD561 NK, and monocyte counts. Results: Of 108 patients, 56 (52%) relapsed at some point during follow-up. Mean annualized relapse rate after alemtuzumab was 0.17 vs 1.67 prior to treatment (equating to a 90% reduction). Of 108 patients, 28 (26%) met the criteria for sustained accumulation of disability. Median time to the lower limit of normal for CD19, CD8, and CD4 was 3, 19.5, and 32 months, respectively. There was no significant difference in the recovery of any cell population between patients with and without disease activity or accumulation of disability after treatment.
Conclusion: This study does not support the use of cell counts as biomarkers for identifying patients at greater risk of active disease following treatment with alemtuzumab. Neurology® 2014;82:2158–2164 GLOSSARY EDSS 5 Expanded Disability Status Scale; IQR 5 interquartile range; LLN 5 lower limit of normal; MS 5 multiple sclerosis; RRMS 5 relapsing-remitting multiple sclerosis.
Editorial, page 2150 Supplemental data at Neurology.org
Alemtuzumab has proven efficacy as a treatment for relapsing-remitting multiple sclerosis (MS). In a phase 2 trial, compared with interferon b-1a, alemtuzumab reduced the risk of relapse and sustained accumulation of disability by more than 70% at 3 years, with sustained efficacy at 5 years.1,2 Two phase 3 trials (CARE-MS I and CARE-MS II) have confirmed efficacy in treatment-naive patients and established superiority over interferon b-1a in patients who continue to relapse despite first-line therapy.2,3 So alemtuzumab was licensed by the European Medicines Agency4 and is entering routine clinical practice in the European Union as a treatment for active MS. Alemtuzumab is a lymphocyte-depleting anti-CD52 monoclonal antibody. Each cycle causes profound pan-lymphocyte depletion, but the relatively infrequent dosing regimen allows reconstitution to occur. The rate and degree of recovery varies with cell type: B cells recover rapidly, whereas T-cell lymphopenia is prolonged, with CD4 and CD8 cells taking 35 and 20 months, respectively, to reach the lower limit of normal.5 During this period of immune reconstitution, 30% of individuals experience thyroid autoimmunity, and 1% develop immune thrombocytopenic purpura; in rare cases, Goodpasture syndrome, autoimmune hemolytic anemia, and autoimmune neutropenia have also been reported.6 We have recently shown that the risk of developing autoimmunity after alemtuzumab is unrelated to rate of T-cell reconstitution but rather reflects the degree to which recovery occurs by expansion of cells that have escaped From the Department of Clinical Neurosciences (O.K.-E., L.A., O.T., A. Compston, A. Coles, J.J.) and the Centre for Applied Medical Statistics, Department of Public Health and Primary Care (R.A.P.), University of Cambridge, UK. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
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depletion rather than thymopoiesis.7 A recently published report,8 however, suggested that peripheral CD4 recovery can be used to predict MS disease activity after treatment, with counts greater than 388.5 3 106 cells/mL at 12 months following therapy identifying patients who are likely to have recurrent disease activity and who may therefore benefit from further treatment. Given the clear clinical implications of this claim, we reassessed this finding in the Cambridge cohort—a larger group of patients in whom the role of alemtuzumab in relapsingremitting MS was originally evaluated and therefore provides prolonged duration of follow-up. METHODS Patients and procedures. All patients had relapsing-remitting MS (RRMS) and had participated in CAMMS223 (a phase 2 randomized controlled trial) and CAMMS 224 or SM3 (both investigator-led, open-label studies). CAMMS223 key eligibility criteria were disease onset within 3 years, at least 2 clinical relapses during the previous 2 years, and a score of 3 or less on the Expanded Disability Status Scale (EDSS). Patients were included in CAMMS 224 and SM3 if they had at least one relapse in the previous year, an EDSS score of 6.0 or less, and disease duration of less than 10 years. Subsequently, all patients entered either CAMSAFE (an investigator-led long-term observational study) or the extension phase of the CAMMS223 trial. The first patient from this cohort was treated on November 22, 1999, with the date for final collection of data January 1, 2013.
Standard protocol approvals, registrations, and patient consents. All studies were approved by a regional ethics board and institutional research committee. All patients gave written informed consent.
Clinical treatment and follow-up protocol. All patients received at least 2 elective cycles of alemtuzumab given annually, with the potential for further cycles if there was clinical or radiologic evidence of ongoing disease activity. Patients were reviewed at 1 and 3 months and then quarterly for the first 2 years after each treatment cycle. For the following 2 years, they were seen biannually and then at least annually thereafter. Patients were also seen whenever a relapse was suspected.
Outcome assessments. For participants in the CAMMS223 study, EDSS scores were determined quarterly in a blinded fashion by a neurologist who also adjudicated possible relapses. The same assessor measured the EDSS of patients in the CAMMS224 and SM3 studies, albeit less frequently. Sustained accumulation of disability was defined as an increase of 1.5 EDSS points from a baseline of 0, or an increase of $1.0 if the baseline was $1.0 confirmed over 6 months. A relapse was defined as new neurologic symptoms attributable to MS lasting .48 hours with an objective change in neurologic examination. Peripheral blood mononuclear cell phenotyping was performed at baseline and then quarterly for the first 36 months and then at least annually (including total lymphocyte count, CD4, CD8, CD19, CD56-NK, and monocyte counts). Brain MRI scans were performed in most patients with a suspicion of active disease prior to retreatment with alemtuzumab. Monthly MRI scans were performed in a subset of patients from the SM3 study. A number of clinically
inactive patients had interval MRI scans to look for subclinical activity and to provide a means for comparison in case of future disease activity.
Statistical analysis. Median time for recovery to the lower limit of normal (LLN) was calculated for each cell subset. All data were categorized depending on the cycle of alemtuzumab treatment. Patients were placed into “active” or “nonactive” groups independent of when an event took place within a particular treatment cycle. Therefore, within each cycle, patients were defined as being relapse-free or relapsing, disability-free or having accumulated disability, or having reached a positive composite endpoint (defined as having relapsed, or accumulated disability, or had an active MRI scan) or a negative composite endpoint based on all 3 outcomes. A subgroup of patients (n 5 91), scanned after treatment, were classified as MRI active or nonactive. To assess differential lymphocyte reconstitution between groups, a linear mixed-effects regression method was undertaken with CD4/CD8/CD19/CD56/monocytes or total lymphocyte count as the outcome variable and relapse/disability/ active MRI/composite score and time point as explanatory variables. A quadratic term (time point squared) was also included due to the observed relationship between time point and outcome. A separate linear mixed-effects model was fitted within each cycle. A continuous autoregressive (order 1) correlation structure was assumed for all models. Model coefficients are presented with 95% confidence intervals and p values. A Fisher exact test was used to assess whether a CD4 count of 388.5 3 106/mL or greater at 12 months predicts disease activity, either clinically or radiologically. The standard 5% significance level was used throughout, and no adjustment made for multiple testing in order to avoid inflating the type II error rate. The linear mixed-effects regression method was implemented in R software8 using the nlme package.9,10 R software was also used to compute Fisher exact tests. All other analyses were performed in GraphPad PRISM (version 5.00 for Windows; www.graphpad.com).
Data were derived from 108 patients, of whom 73 (67.6%) were female. The median follow-up from first treatment was 99 months (interquartile range [IQR] 74.75– 117.25). The mean age of patients at first treatment with alemtuzumab was 32.8 years (SD 7.99). The median EDSS at baseline was 3.0 (IQR 1.5–4.75). Mean relapse frequency prior to treatment was 1.7 relapses per annum (SD 0.81).
RESULTS Study population characteristics.
Lymphocyte reconstitution. As previously reported,5,8,11
treatment with alemtuzumab led to profound panlymphocyte depletion, followed by differential recovery. CD19 lymphocytes reached the LLN most rapidly, with a median recovery time of 3 months (IQR 3–6). The intervals for CD8 and CD4 lymphocytes were median times of 19.5 (IQR 10–34.5) and 32 (IQR 21.75–41) months, respectively. Clinical outcomes. The total number of patients who experienced at least 1 relapse during the follow-up period was 56 (51.85%). The mean relapse frequency posttreatment was 0.17 relapses per annum, equating to an 89.8% reduction in the annualized relapse rate Neurology 82
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compared to pretreatment. Twenty-eight patients (25.9%) met the definition for sustained accumulation of disability. Ninety-one patients had an MRI scan: 16 individuals (17.6%) had an active scan (new T2/ enhancing lesions) at some point during the follow-up period (see tables e-1 and e-2 on the Neurology® Web site at Neurology.org for a detailed breakdown of each alemtuzumab cycle). Association of peripheral mononuclear cell subsets with disease activity. Relapse. Within each treatment cycle,
there was no difference in the number of CD4 T cells, CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those with and without clinically defined relapses (figure 1 for cycles 1-3, figure e-1 for cycle 4, and table 1; data not shown for NK cells and monocytes). Using the Fisher exact test, we found no association between a CD4 count of .388.5 cells 3 106/mL at 12 months and risk of relapse (p 5 0.28). Given the possibility that relapses within the first few months of treatment may be due to lymphocytes that have already entered the CNS,12 we looked at timing of relapses following cycle 2 (chosen as it is the most informative cycle in terms of patient numbers, number of relapses, and length of follow-up). Only 3 out of 106 patients relapsed within 2 months of treatment, Figure 1
and of these, 2 went on to have additional relapses within cycle 2, leaving only one patient who was potentially misclassified using our method. Given this, we are confident in our conclusions. MRI activity. Within each treatment cycle, there was no difference in the number of CD19 B cells, CD56 NK cells, or monocytes between those with and without active MRI scans (figure 2 for cycles 1-3, figure e-1 for cycle 4, and table 1; data not shown for NK cells and monocytes). CD4 cells (p 5 0.016) and CD8 cells (p 5 0.008) were found to be higher (on average by 0.146 and 0.125 3 109/mL cells, respectively) in the active MRI group (n 5 5) vs the inactive group (n 5 28) within treatment cycle 3. No difference was found in any other treatment cycle; indeed, in cycles 1 and 2 (the most informative periods numerically), the trend was in the opposite direction (figure 2, table 1). Using the Fisher exact test, we found an association between a CD4 cell count of .388.5 3 106/mL at 12 months and the risk of having an active MRI scan (overall p 5 0.02). However, further analysis of this result demonstrated that the difference was driven by patients within cycle 3 (p , 0.0001), with no difference observed within cycles 1 (p 5 1.0), 2 (p 5 0.91), or 4 (p 5 0.05). Disability accumulation. Within each treatment cycle, there was no difference in the number of
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without on-study relapses
Patients were defined as having relapsed (shown in red) if they developed neurologic symptoms attributable to multiple sclerosis lasting .48 hours with an objective change in neurologic examination in the absence of infection. Cell units are 3109/L. Error bars indicate SD. 2160
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Table 1
Comparison of CD41, CD81, and CD191 counts after each cycle of alemtuzumab in those with and without active disease CD4 Cycle
Relapse
MRI
Disability
Composite
CD8
Coefficient
95% CI
CD19
p Value
Coefficient
95% CI
p Value
Coefficient
95% CI
p Value
1
0.003
0.028 to 0.034
0.859
20.020
20.069 to 0.030
0.438
0.025
20.026 to 0.077
0.330
2
20.029
20.075 to 0.017
0.209
20.011
20.051 to 0.028
0.577
0.040
20.007 to 0.087
0.095
3
20.009
20.083 to 0.065
0.806
0.037
20.017 to 0.090
0.173
0.005
20.069 to 0.079
0.901
4
20.042
20.333 to 0.248
0.741
20.046
20.255 to 0.163
0.620
20.031
20.376 to 0.314
0.839
1
20.053
20.117 to 0.012
0.104
20.071
20.168 to 0.027
0.144
0.011
20.124 to 0.146
0.865
2
20.007
20.094 to 0.080
0.873
20.026
20.116 to 0.063
0.558
0.010
20.086 to 0.106
0.831
3
0.146
0.029 to 0.264
0.016
a
0.125
0.035 to 0.214
0.008
0.029
20.101 to 0.159
0.653
4
0.068
20.239 to 0.375
0.606
0.107
20.134 to 0.348
0.319
20.228
20.588 to 0.131
0.171
1
20.063
20.103 to 20.024
0.002a
20.008
20.076 to 0.060
0.813
0.042
20.027 to 0.111
0.226
2
20.041
20.099 to 0.016
0.154
20.009
20.057 to 0.039
0.709
0.005
20.055 to 0.065
0.861
3
20.028
20.114 to 0.058
0.522
20.022
20.084 to 0.040
0.473
20.001
20.087 to 0.084
0.977
4
20.204
20.546 to 0.139
0.203
20.104
20.374 to 0.166
0.392
20.054
20.508 to 0.399
0.784
1
20.019
20.047 to 0.009
0.179
20.004
20.050 to 0.041
0.860
0.013
20.033 to 0.060
0.571
2
20.028
20.074 to 0.018
0.233
0.002
20.035 to 0.039
0.923
0.021
20.025 to 0.068
0.367
3
20.022
20.095 to 0.051
0.550
0.021
20.032 to 0.074
0.432
0.004
20.069 to 0.077
0.915
4
20.285
20.548 to 20.023
0.037a
20.184
20.390 to 0.022
0.073
20.107
20.548 to 0.335
0.587
a
Abbreviation: CI 5 confidence interval. For each cycle, patients were defined as active or not based on clinical relapse, MRI activity, disability acquisition, and the composite endpoint. A linear mixed-effects regression method was used with CD41, CD81, CD191, CD561 NK cells, or monocytes as the outcome variable, and with relapse, disability, MRI activity, or composite score and time point as explanatory variables. A quadratic term (time point squared) was also included due to the observed relationship between time point and outcome. A separate linear mixed-effects model was fitted within each cycle. A continuous autoregressive (order 1) correlation structure was assumed for all models. Model coefficients are presented with 95% CIs and p values are shown for CD41, CD81, and CD191 cells (other subpopulations are reported in the text). a 5% significance level reached.
CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those with and without accumulation of disability (figure 3 for cycles 1-3, figure e-1 for cycle 4, and table 1). CD4 T cells were found to be lower in patients who accumulated disability in cycle 1 (adjusted mean difference across the cycle 0.063 3 109; p 5 0.002). No difference was found in any other treatment cycle, although the trend was in the same direction (figure 3, table 1). Composite. Within each treatment cycle, there was no difference in the number of CD8 T cells, CD19 B cells, CD56 NK cells, or monocytes between those who did and did not reach the composite end point (table 1; data not shown for nonlymphocyte cell populations). Within cycle 4, CD4 T cells were found to be lower in patients who met the composite end point compared to those who did not. No difference was found in any other treatment cycle (table 1). Using a much larger cohort and more prolonged follow-up, we fail to confirm the claim that accelerated CD4 T cell recovery after treatment is a biomarker for recurrent MS disease activity following lymphocyte depletion with alemtuzumab. We also find no evidence that a CD4 T-cell count DISCUSSION
of greater than 388.5 3 106 cells/mL at 12 months has utility in selecting a group of patients who may benefit from more intensive monitoring or perhaps even prophylactic repeat dosing. There are a number of differences between our work and the previous report.8 First, our cohort is larger (108 vs 56 patients) with a longer duration of follow-up (median follow up of 99 months [IQR 74.75–117.25] vs 55 months [IQR 24–115]). Although both studies selected patients with active RRMS, baseline MS disease activity was somewhat higher in the previous study (annualized relapse rate of 2.6 SD 0.9 vs 1.7 SD 0.8); however, few of their patients experienced disease activity post alemtuzumab (probably reflecting their shorter follow-up): only 8/56 experienced a clinical relapse, with a further 4 patients showing MRI disease activity alone; this small number of data points makes the study susceptible to extreme outliers. Unlike the previous study, we did not perform routine MRI brain scans at month 24. As a consequence, analysis of MRI outcome is based on data from fewer patients (19 for cycle 1, 59 for cycle 2, 33 for cycle 3, and 10 for cycle 4); this is a limitation of our study. However, these differences do not explain why our 2 studies have reached opposite conclusions; this is best Neurology 82
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Figure 2
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without on-study active MRI scans
Patients were defined as having an active MRI scan (shown in red) if they had acquired new T2 lesions or enhancing lesions. Cell units are 3109/L. Error bars indicate SD.
explained by weaknesses in their statistical methods. First, they did not account for repeated treatments; only cell counts from the most recent alemtuzumab dose were analyzed: post cycle 2 CD4 counts for those who remain in remission were compared to post cycle 3 counts for those with active disease prompting redosing and CD4 counts post cycle 3 were then correlated with disease activity prior to cycle 3 and used to predict an event that had already occurred. This method assumes that reconstitution is identical after each round of treatment, representing a major limitation of their study. We controlled for this bias by looking at reconstitution and disease activity after and within each treatment cycle. Due to complexity of the analysis, the timing of the event within each cycle is still not accounted for. Secondly, the previous report compared mean cell counts at multiple time points using Student t tests, or Mann-Whitney U when non-normally distributed, without taking into account multiple nonindependent observations per patient (an individual’s CD4 count at month 12 is not independent of the month 9 count, and so on). Furthermore, p values were not corrected for multiple comparisons, of which there were many, so it is likely that some of the statistically significant results occurred by chance; when we repeated the analysis using our data, no p value survived correction (data not shown). 2162
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The wish to identify a biomarker for recurrent disease activity after alemtuzumab is to be welcomed. This would reduce the need to monitor patients at low risk of relapse and allow the preemptive treatment of highrisk patients. Although CD4 counts may be an attractive candidate—they are readily measurable and T cells are undoubtedly involved in disease pathogenesis—given the complex nature of the immune system, it is not surprising that peripheral CD4 counts alone do not predict CNS inflammation (indeed, it is known that selective anti-CD4-depleting therapies do not suppress disease activity in MS13). Also, after alemtuzumab treatment, composition of the circulating immune repertoire is radically altered. For example, for at least 6 months following each cycle, the CD4 T-cell pool is dominated by memory cells, particularly those with a regulatory phenotype (CD41CD45RA-CD35hiFoxP31IL-7Rlo).14 Self evidently, investing confidence in a single measure of a major cellular constituent of peripheral blood disregards the complexity of the immunopathogenesis of MS and is misplaced. Arguably, our data do not prove or disprove whether long-term disease stability is associated with lower CD4 counts, as patients with clinical or radiologic evidence of disease activity are automatically retreated. However, it is our position that peripheral
Figure 3
Comparison of CD41, CD81, and CD191 cell counts after each cycle of alemtuzumab in patients with and without acquisition of disability
Patients were deemed to have met the definition of sustained accumulation of disability if their Expanded Disability Status Scale increased by $1.5 points from a baseline of 0, or by $1.0 points from a baseline of $1.0. Patients meeting this definition are shown in red. Cell units are 3109/L. Error bars indicate SD.
CD4 counts have no utility in predicting MS disease activity after alemtuzumab, and we strongly advise neurologists against using them to personalize treatment protocols. In particular, CD4 counts should not be used as a marker of the need for preemptive retreatment, thereby exposing patients to potential risk.1–3 Our cautionary message, refuting the claims of the previous report,8 is timely since alemtuzumab will soon be entering the clinic as a treatment for active RRMS. AUTHOR CONTRIBUTIONS Dr. Onajite Kousin-Ezewu designed and conducted the study, analyzed data in the study, interpreted data in the study, and drafted and revised the manuscript. Dr. Laura Azzopardi conducted the study and interpreted data in the study. Mr. Richard Parker analyzed data in the study. Dr. Orla Tuohy conducted the study. Prof Alastair Compston revised the manuscript. Dr. Alasdair J. Coles designed the study and revised the manuscript. Dr. Joanne L. Jones designed the study, analyzed data in the study, interpreted data in the study, and revised the manuscript.
STUDY FUNDING No targeted funding reported.
DISCLOSURE O. Kousin-Ezewu, L. Azzopardi, R. Parker, and O. Tuohy report no disclosures relevant to the manuscript. A. Compston reports receiving consulting fees, lecture fees, and grant support from Genzyme, and lecture fees from Bayer Schering Pharma, and has consulted for Lundbeck, all on behalf of the University of Cambridge. A. Coles reports receiving consulting and
lecture fees from Genzyme, lecture fees from Merck Serono, and research support paid to his institution from Genzyme. J. Jones reports receiving consulting fees and lecture fees from Bayer Schering Pharma and lecture fees from Genzyme. Go to Neurology.org for full disclosures.
Received August 24, 2013. Accepted in final form February 7, 2014. REFERENCES 1. Coles AJ, Compston DA, Selmaj KW, et al. Alemtuzumab vs. interferon beta-1a in early multiple sclerosis. N Engl J Med 2008;359:1786–1801. 2. Cohen JA, Coles AJ, Arnold DL, et al. Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial. Lancet 2012;380:1819–1828. 3. Coles AJ, Twyman CL, Arnold DL, et al. Alemtuzumab for patients with relapsing multiple sclerosis after diseasemodifying therapy: a randomised controlled phase 3 trial. Lancet 2012;380:1829–1839. 4. European Medicines Agency: EPAR summary for the public: Lemtrada (alemtuzumab). Available at: http://www.ema. europa.eu/docs/en_GB/document_library/EPAR_-_Summary_ for_the_public/human/003718/WC500150523.pdf. Accessed November 22, 2013. 5. Hill-Cawthorne GA, Button T, Tuohy O, et al. Long term lymphocyte reconstitution after alemtuzumab treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry 2012; 83:298–304. 6. Coles AJ, Fox E, Vladic A, et al. Alemtuzumab more effective than interferon-1a at 5-year follow-up Neurology 82
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Accelerated lymphocyte recovery after alemtuzumab does not predict multiple sclerosis activity Onajite Kousin-Ezewu, Laura Azzopardi, Richard A. Parker, et al. Neurology 2014;82;2158-2164 Published Online before print May 16, 2014 DOI 10.1212/WNL.0000000000000520 This information is current as of May 16, 2014 Updated Information & Services
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Supplementary material can be found at: http://www.neurology.org/content/suppl/2014/05/16/WNL.00000 00000000520.DC1.html http://www.neurology.org/content/suppl/2014/06/15/WNL.00000 00000000520.DC2.html
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